Sensitivity to benzo(a)pyrene of epithelial and mesenchymal cells of embryonic lungs of mice susceptible (line A) and resistant (line C57BL) to lung blastomogenesis

Proliferative activity of alveolar (EA) and bronchial (EB) epithelial cells, mesenchymal cells of explants (ME) and mesenchymal cells migrated on the cellulose filter (MF) was studied autoradiographically in the normal cultures and after treatment with benz(a)pyrene (BP). Labeling index (LI) of cells from the treated explants was higher or lower than in normal cultures, or did not differ from normal values. The effect of the treatment depended on BP dose, mouse strain, and the type of cells. Epithelial cells, especially EB, and ME cells from embryonic lungs of A mice were very sensitive to growth-stimulating effect of BP. In treated cultures from C57BL mice LI increased only in EB cells, however, unlike A mice, the effect inversely correlated with BP doses. Proliferative activity of EA, ME and MF cells was lower than in untreated explants from C57BL, the effect of treatment increasing with higher BP doses. The importance of local cell-tissue factors, namely epithelial-mesenchymal interactions, and the role of lung mesenchyma in the realization of genetically determined sensitivity to spontaneous and induced lung blastomogenesis in C57BL and A mice are discussed.     

Increasing the toxic effect of pulmonary carcinogens by destruction of epithelial-mesenchymal interaction in explants of embryonic respiratory tract of mice

The explants from 17-day old embryos of A and C57BL mice were used for long-term organ cultures. The following series of explants were studied: 1) the intact control explants; 2) the explants treated by NMU; 3) the explants treated by BP; 4) the explants isolated from mesenchyma (M); 5) the explants isolated from M and treated by NMU; 6) the explants isolated from M and treated by BP. The survival of explants treated by NMU or BP did not differ from the intact control explants (p less than 0.1). The removal of M decreased the survival only in the explants from the distal RT of A mice (p 0.01). The survival of explants isolated from M and treated by NMU or BP was significantly lower than it was in intact explants (p less than 0.01-0.001). Thus, the destruction of epithelial-mesenchymal interaction induced by removal of M can modulate the toxic effect of pulmonotropic carcinogens.     

Characteristics of cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung blastomogenesis

Linear differences were discovered in the quantitative ratio and proliferative activity of the cells of the epithelium and mesenchyma of organ cultures of the normal embryonal pulmonary tissue of mice resistant (C57BL) and predisposed (A) to lung blastomogenesis. In A mouse embryos, the index of the cell labels of the bronchiolar epithelium and fibroblast-like cells of the mesenchyma which migrate on the surface of the membrane filter was several times higher than that in C57BL mice. The number of the mesenchymal cells capable of migration in A mouse explants was essentially greater, that of the mesenchymal cells being in a direct contact with the epithelium of alveoloid and bronchiolar structures was, on the contrary, lower than in C57BL mouse explants. It is suggested that the differences described, particularly epithelial-mesenchymal interactions, are of importance for realization of the genetically determined sensitivity of these mice to spontaneous and induced lung blastomogenesis, which manifests as early as during prenatal ontogenesis.     

Partial removal of the mesenchyma disrupts the growth and differentiation of the epithelium in respiratory tract explants from strain A and C57BL mouse embryos

Differentiation of respiratory epithelium (E) of 17-day old embryos of A and C57BL mice after partial removal of mesenchyme (M) was studied in organ culture. In control explants, tissue-specific growth and differentiation of epithelium were observed during long-term culturing. Ih both mouse strains, partial removal of mesenchyme prevented the development of alveolar-like structures in explants of distal part of respiratory tract. In most explants of proximal part of respiratory tract (65.1% in A mice and 85.7% in C57BL mice) with partially removed mesenchyme, we found atypical epithelial structures and foci of poorly differentiated cells with high proliferation rate.     

Dynamics of proliferative activity of cultures of organotypic epithelial-mesenchymal recombinants from embryonic lungs of intact and urethane-receiving mice]

The dynamics of proliferative activity of cells was studied in the cultures of organotypic recombinants obtained from embryonic lung epithelium (E) and mesenchyma (M) of the intact and treated transplacentally by urethane mice (strain A). The labelling index (LI) of E and M in the aggregates obtained from treated embryonic lungs (EtMt) was significantly higher than LI in the aggregates from intact embryonic lungs (EiMi) in all days of cultivation (4-7-14). M from the treated embryonic lungs stimulated LI of the intact E (EiMt) but M from the intact embryonic lungs decreased LI of the treated E (EtMi).     

Allogenic inhibition of the stem hematopoietic cells in the bone marrow of adult mice and in the embryonal liver]

The maternal effect was shown to influence the degree of allogenic inhibition of stem hemopoietic cells of the embryonic liver and adult bone marrow in CBA and C57Bl/6 mice. The display of allogenic inhibition of stem cells of the embryonic liver and adult bone marrow proved to be similar in C57Bl/6 mice and dissimilar in CBA.     

Transplacental effect of methylcobalamine on the growth of embryonic mouse kidney tissue in organotypic cultivation

Transplacental growth-stimulating effect of the Cbl-coenzyme methylcobalamine (MeCbl) was revealed in organ cultures of embryonic kidney tissue in DBA/2 mice. MeCbl improved the survival of embryonic kidney explants and decreased the incidence of degenerative damage in cultures. The frequency of regeneration development was 30%. Hyperplastic epithelial changes were much more marked after transplacental MeCbl administration (71.8%), than in the control (9.0%).     

Morphogenetic characteristics of the aggregates formed in epithelial and mesenchymal recombinations of the lungs from intact and urethane-exposed mouse embryos

A study was made of the morphogenesis of organotypic aggregates obtained by epithelial mesenchymal recombinations from the lungs of embryonic mice, intact and treated with urethane. Normal growth and differentiation of organotypic structures were observed in long-term cultures of aggregates obtained by recombinations of the lung epithelium (E) and mesenchyma (M) from intact (i) embryonic mice (EiMi). Hyperplasia and squamous-cell metaplasia (with or without keratinization) of the epithelium were found in aggregates obtained from E and M of the treated mouse embryos (EtMt) and in aggregates obtained by recombinations of lung E and M from intact and treated embryos (EtMi, EiMt). The data obtained suggest that the alterations in epithelial mesenchymal interactions are of great significance for transplacental lung blastomogenesis and that the mesenchymal lung cells play an important part in mediation of the transplacental carcinogenous effects on epithelial target cells via subsequent epithelial mesenchymal tissue interactions.     

Trypanosoma cruzi: explant organ cultures from mice with chronic Chagas' disease

Explants of 13 different organs obtained from C3H/HEN, Swiss-Webster, and C57Bl/6 mice chronically infected with Trypanosoma cruzi (Y strain) were cocultivated with mouse embryo fibroblasts to determine the organs that contain T. cruzi during the chronic infection. Explant cultures frequently yielded T. cruzi as late as 12 months after infection. Spleen and skeletal muscle were most frequently positive; heart cultures were rarely positive in any mouse strain. C3H/HEN mice had significantly more cultures positive than Swiss-Webster mice, as expected from relative susceptibility of C3H/HEN mice to acute infection. In contrast, C57Bl/6 mice, relatively resistant to acute infection, had significantly more cultures positive at 12 months of infection than Swiss-Webster mice. Also, C57Bl/6 mice had a significant increase in the number of positive cultures at 12 months of infection compared to 6 months of infection. These results show that organisms can be recovered routinely from some tissues during the chronic infection, that murine susceptibility to infection should differentiate between acute and chronic infection, and that C57Bl/6 mice may lose control of infection during the chronic infection.     

Potentiation by caffeine of the frequencies of micronuclei induced by mitomycin C and cyclophosphamide in young mice

Employing the micronucleus test in mouse bone marrow and in fetal mouse liver, the possible clastogenicity of caffeine as well as its influence on MMC- and CP-induced micronucleus levels were studied. The treatment of male and female C57Bl or BDF1 (C57Bl x DBA2) mice with caffeine (1 or 3 x 50 mg/kg and 100 mg/kg, s.c.) had no clastogenic effect in mouse bone marrow or in the fetal livers and maternal bone marrow when pregnant mice were injected with caffeine on day 16-17 of gestation. MMC (2.0 mg/kg, i.p.) increased up to 10-30-fold the number of MNPCEs in bone marrow compared to a 3-7 fold elevation of MNPCEs in fetal liver. A similar effect was also established in pregnant mice treated with CP (30 mg/kg, i.p.). No significant sex differences in spontaneous and MMC- or CP-induced MNPCEs levels were established in C57Bl and BDF1 mice. However, a significantly higher spontaneous rate of MNPCEs as well as a better-expressed responsiveness to the clastogenic activity of MMC and CP were established in C57Bl compared to BDF1 mice. The pregnancy had no effect on MMC- or CP-induced clastogenicity although a tendency to a decreased sensitivity to the damaging activity of MMC seemed to be detected in pregnant C57Bl mice compared to virgin female animals. The combined treatment of mice with caffeine (3 x 100 mg/kg) and MMC or CP caused an up to 45-49% potentiation of clastogenesis in the bone marrow of male, female and pregnant female C57Bl and BDF1 mice but not in fetal mouse livers.     

Poorer NF-kappa B signaling by microfilariae in macrophages from BALB/c mice affects their ability to produce cytotoxic levels of nitric oxide to kill microfilariae

Upon activation with microfilariae (mf), macrophages from C57Bl/6 mice showed higher nuclear factor-kappa B (NF-kappa B) but lower activating protein 1 DNA-binding activity as compared to BALB/c macrophages. The C57Bl/6 macrophages produced cytotoxic levels of nitric oxide (NO) to kill Setaria cervi mf as compared to BALB/c macrophages. Inhibition of the NF-kappa B signal by pyrrolidine dithiocarbamate (PDTC) blocked NO production and microfilaricidal activity of C57Bl/6 macrophages and inclusion of the exogenous NO generator (SNP) in the PDTC treated C57Bl/6 macrophage cultures induced mf cytotoxicity. These results underscore that the NF-kappa B signal (induced in response to mf) is important for the NO-mediated microfilaricidal activity of macrophages.     

Sensitivity of embryonal lungs of mice, rats and humans to exposure to nitrosomethylurea in organ cultures

With the direct action of nitrosomethylurea (NMU) in a concentration of 0.05 mg/ml on the organic cultures of the embryonic lung of mice of the A line, Wistar rats and man they developed a different degree of degenerative changes and hyperplastic epithelial proliferates. A toxic effect prevailed in the cultures at the initial experimental periods. The most sensitive to the toxic action of NMU was the lung tissue of rat embryos, and the least--of mice. The incidence of hyperplastic proliferates was, on the contrary, the greatest in the cultures of mouse lungs, and the least--of rat lungs. The sensitivity of the embryonic lungs of the man and rodents to the toxic action of NMU in repeated administration into the nutrient medium diminished during the cultivation. There was an increase of survival of the experimental cultures in comparison with the intact control.     

Immunologgical self-tolerance in allophenic and embryo-aggregated mice

Allophenic mice, supposedly containing almost equal numbers of cells derived from embryos of mouse strains C57Bl and FVB, were shown in a recent paper to grow the B16 melanoma, a long transplanted tumor of C57Bl origin, much better than did mice of either the parental C57Bl strain or the C57Bl × FVB F1 hybrid. Mice containing smaller proportions of C57Bl cells rejected the tumor. A reconsideration of these suprising data, in light of the current literature, suggests that the better growth of the tumor in the 50-50% allophenics than in the C57Bl parental strain was almost certainly caused by the tumor stimulation engendered by a weak anti-C57Bl immune reaction in the overtly healthy allophenic mice.     

Cancer immunotherapy by immunosuppression

Allophenic mice, supposedly containing almost equal numbers of cells derived from embryos of mouse strains C57Bl and FVB, were shown in a recent paper to grow the B16 melanoma, a long transplanted tumor of C57Bl origin, much better than did mice of either the parental C57Bl strain or the C57Bl × FVB F1 hybrid. Mice containing smaller proportions of C57Bl cells rejected the tumor. A reconsideration of these suprising data, in light of the current literature, suggests that the better growth of the tumor in the 50-50% allophenics than in the C57Bl parental strain was almost certainly caused by the tumor stimulation engendered by a weak anti-C57Bl immune reaction in the overtly healthy allophenic mice.     

Experimental model for studying the participation of bone marrow cells in antibody formation

Participation of bone marrow cells in the production of IgM antibody forming cells (AFC) in the primary immune response to sheep red blood cells (SRBC) in C57Bl/6 and BDA/2 mice was studied. The animals of this line differed in sensitivity to preoral benz(a)pyren (BP) injection. After BP injection a toxical injury of bone marrow cells was observed for two days in DBA 2 mice but was not marked in C57Bl/6 mice. In the former it was followed by a 10-fold decrease of IgMAFC, while no profound changes were noticed in the immune response of the latter. A new model is offered for the evaluation of bone marrow cell participation. A suggestion is made concerning some connection of immunodepression in the bone marrow with the change of the stem hemopoietic precursor differentiation.     

Protection of mice against influenza virus infection: Enhancement of nonspecific cellular responses by Corynebacterium parvum

Groups of C57Bl/6J, BALB/c, BALB/c nu⁺/nu⁺ mice, inoculated intranasally with Corynebacterium parvum (350 μg/mouse) were protected from death by an otherwise lethal dose of influenza virus, A/WSN (H1N1) inoculated 3 days later. The lungs of C. parvum-trented, virus-infected C57Bl/6J, BALB/c, or BALB/c nu⁺/nu⁺ mice contained significantly less infectious virus than did controls, and this reduction was apparent as soon as 24 hr after virus inoculation. The maximum protective effect correlated with increased lung interferon levels. C. parvum treatment caused an increase in the lung cell number which was in part due to a large increase (ca. 10-fold) in macrophage content, and the natural killer cell activity was also enhanced, though not as markedly as occurred 3 days after infection. Most (>85%) of the resident macrophages in normal lungs were susceptible to infection by virus (as indicated by hemadsorption), whereas most of those recovered from the lungs of C. parvum-treated mice resisted infection. Despite the increase in macrophage content, the level of specific immune responses to infection, such as cytotoxic T-cell activity, DTH reaction, and antihemagglutinin antibody, remained unchanged by C. parvum treatment so that the major if not only effect of this treatment was on the level of the less-specific components of the immune system.     

Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs

The sensitivity to the fibrosis-inducing effect of bleomycin varies considerably from species to species, the reasons for which are unknown. The variability of the response in different strains of mice is well documented. Recent evidence indicates that the upregulated expression of cytokines and cytokine receptors may be involved. We evaluated the expression pattern of some cytokines and their receptors in C57Bl/6J bleomycin-sensitive and Balb/C bleomycin-resistant mice. Animals from both strains received, under ether anesthesia, either saline (50 microl) or bleomycin (0.1 U/50 microl) intratracheally. At various times after the treatment, the lungs were analyzed for cytokines and cytokine receptors by histochemistry and their mRNA by RNase protection assay. A significantly increased expression of TNF-alpha and IL-1beta was observed in both strains. However, an upregulated lung expression for TNF-alpha and IL-1 receptors was observed in C57Bl/6J-sensitive animals only. This profile is evident from 63 h onward. In addition to TNF-alpha, bleomycin administration also resulted in the upregulated expression of TGF-beta in the lungs of both strains at 8 h and in an enhanced expression of TGF-beta receptors I and II in C57Bl/6J mice only. The upregulation of TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13, and IL-13 receptor-alpha (at 8 h after bleomycin) and followed by an upregulation of gp130 and IL-6. The difference we observed in the cytokine receptor profile may offer an additional explanation for the different fibrogenic response of the two mouse strains to bleomycin.     

Estrogen Alters MPTP-Induced Neurotoxicity in Female Mice: Effects on Striatal Dopamine Concentrations and Release

Abstract: The effects of estrogen on MPTP-induced neurotoxicity of the nigrostriatal dopaminergic system were examined in C57Bl and CD-1 mice. Ovariectomized mice with and without estrogen were treated with MPTP or its vehicle. The effects of these treatments on striatal dopamine concentrations and l -DOPA-stimulated dopamine and l -3,4-dihydroxyphenylacetic acid (DOPAC) release in vitro were determined. Dopamine concentrations of C57Bl mice receiving estrogen before MPTP were significantly greater than those of non-estrogen-treated MPTP mice as well as estrogen-treated mice receiving the MPTP vehicle. Dopamine concentrations of the CD-1 mice did not differ with these treatments. l -DOPA-evoked dopamine release values of estrogen-treated C57Bl mice were significantly increased compared with non-estrogen-treated mice. No such differences were observed in the MPTP-treated C57Bl mice. DOPAC release rates were similar to that of dopamine in these C57Bl mice. In the CD-1 mice estrogen also produced a significant increase in l -DOPA-evoked dopamine release; however, this response was unaltered by MPTP treatment. A significant increase in l -DOPA-evoked DOPAC output was obtained only for estrogen-treated CD-1 mice. Both strains show very similar responses to the estrogen treatment, but differential responses of dopamine release to l -DOPA between the C57Bl and CD-1 mice were obtained with regard to the interactive effects of estrogen and MPTP. Our results suggest that in addition to its role as modulator, estrogen may also function as a neuroprotectant against MPTP neurotoxicity of the nigrostriatal dopaminergic system in the C57Bl mouse.     

B cell response during infection with the MAT a and MAT alpha mating types of Cryptococcus neoformans

In the present study, we compared the B cell response of BALB/c and C57Bl/6 mice during Cryptococcus neoformans infection. This response was investigated using virulent serotype D forms of mating types alpha and a (MAT alpha and MAT a). C57Bl/6 mice showed massive (mainly cerebral) infection by both types, while BALB/c were resistant to infection. Some resistance of C57Bl/6 mice was induced by previous immunization with the capsular polysaccharide from MAT alpha. Passive immunization of C57Bl/6 mice with purified antibody (Ab) obtained from capsular polysaccharide-immunized mice also increased resistance to infection. Both mouse strains showed comparable low IgM response to the capsular polysaccharide from MAT alpha, and only C57Bl/6 mice produced IgM to the polysaccharide of MAT a. Comparable levels of different immunoglobulin (Ig) isotypes against capsular components of MAT alpha and MAT a were detected, and the response of C57Bl/6 mice was higher when compared to that of BALB/c mice. FACS analysis indicated an increase in the percentage of a high-granulosity (side-scatter) splenic subpopulation and in the percentage of splenic Gr-1+ cells in infected C57Bl/6 mice. In addition, the percentage of follicular splenic B cells was decreased after C. neoformans infection of C57Bl/6 mice. This response was more pronounced when we investigated infection induced by the MAT a mating type. Taken together, our results indicate that capsular polysaccharide derived from MAT alpha and MAT a types of C. neoformans have a stimulatory effect upon B cells but that there is no correlation between resistance of BALB/c mice and Ab production. However, the increase in resistance of C57Bl/6 mice parallels the production of Abs and a major change in splenic cell populations.     

Rac1 mutations produce aberrant epithelial differentiation in the developing and adult mouse small intestine

The mouse small intestinal epithelium undergoes continuous renewal throughout life. Previous studies suggest that differentiation of this epithelium is regulated by instructions that are received as cells migrate along crypt-villus units. The nature of the instructions and their intracellular processing remain largely undefined. In this report, we have used genetic mosaic analysis to examine the role of Rac1 GTPase-mediated signaling in controlling differentiation. A constitutively active mutation (Rac1Leu61) or a dominant negative mutation (Rac1Asn17) was expressed in the 129/Sv embryonic stem cell-derived component of the small intestine of C57Bl/6-ROSA26<->129/Sv mice. Rac1Leu61 induces precocious differentiation of members of the Paneth cell and enterocytic lineages in the proliferative compartment of the fetal gut, without suppressing cell division. Forced expression of the dominant negative mutation inhibits epithelial differentiation, without affecting cell division, and slows enterocytic migration along crypt-villus units. The effects produced by Rac1Leu61 or Rac1Asn17 in the 129/Sv epithelium do not spread to adjacent normal C57Bl/6 epithelial cells. These results provide in vivo evidence that Rac1 is involved in the import and intracellular processing of signals that control differentiation of a mammalian epithelium.